In vivo generation of hybrids between different species of RNA phages

Abstract

Hybrids between different species or genera of the single-stranded RNA coliphages have not been found in nature. Here, it has been shown that viable hybrids between different phage species can easily be generated in the laboratory by in vivo recombination. cDNA of species I phage MS2 located on a plasmid and lacking part of its 5h untranslated leader (5h UTR) was complemented with another plasmid carrying the 5h half of the genome of fr, a species I phage, or of KU1, a species II representative with low sequence similarity. When the two plasmids were present in the same cell there was spontaneous production of hybrid phages. Interestingly, these hybrids did not arise by a double or single crossover that would replace the missing MS2 sequences with those of fr or KU1. Rather, hybrids arose by attaching the complete 5h UTR of fr or KU1 to the 5h terminus of the defective MS2 phage. Several elements of the 5h UTR then occurred twice, one from KU1 (or fr) and the other from MS2. These redundant elements are in most cases deleted upon evolution of the hybrids. As a result, the 5h UTR of KU1 (or fr) then replaced that of MS2. It was earlier shown that this 5h UTR could assume two alternating structures that facilitated transient translation of the proximal maturation gene. Apparently, this timer function of the 5h UTR was exchangeable and could function independently of the rest of the genome. When hybrids were competed against wildtype, they were quickly outgrown, probably explaining their absence from natural isolates.

RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts

“…According to a copy-choice model, prior to the actual switch the replicase stalls or dislodges from its template (21,28,36), which may occur at the 5Ј end of the template (47,49), at a template break (39), or at the nascent strand stem-loop structure. The latter is analogous to rho-independent transcription termination (46,50), although it may be distinct from a dislodging RdRp in a copy-choice model.…”